Gottesman Project Title Summary We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia colias a model system. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to present RpoS to the ClpXP protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. Deletion analysis of the rssB gene demonstrates that both the N-terminus and C-terminus of the protein are necessary for degradation of RpoS. Deletions of the C-terminus lead to sequestration of substrate in an inactive but stable form. Specific interactions between the deleted derivatives, ClpX, and RpoS will be studied in vitro. In addition to regulation of RpoS degradation, RpoS translation is positively regulated by two small RNAs, DsrA and RprA. Regions within each of these small RNAs can pair with the RpoS leader mRNA. This disrupts the secondary structure of the RpoS leader, allowing translation. The promoter of dsrAis regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation can be fully relieved by a single base pair change in the -10 region of the promoter, suggesting that unusual promoter structures may be mediating temperature regulation. RprA, identified as a suppressor of dsrAmutants, is regulated by the two component RcsC and RcsB regulators. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein; they are activated by cell surface stress. When they are activated, RpoS synthesis increases in an RprA-dependent fashion. Comparison between the two small RNAs allows development of a consensus sequence necessary for positive regulation of RpoS, as well as demonstrating how multiple environmental signals can be sensed to modify RpoS availability. A collaboration with the laboratory of Dr. Gisela Storz in NICHD and Dr. Carsten Rosenow at Affymetrix to identify other small RNAs in E. coli was recently completed. We identified conserved intergenic regions and scanned a number of these both with microarray analysis and Northern blots to identify 17 novel small RNAs (sRNAs) and five novel mRNAs encoding short ORFs. In addition, we found that many of these sRNAs bind to the RNA binding protein, Hfq, essential for DsrA action and RpoS translation. Our approaches can be applied to searches for small RNAs in other organisms. One of the newly identified sRNAs, RyhB, negatively regulates translation of sdh, encoding succinate dehydrogenase. RyhB synthesis in turn is negatively regulated by the Fe-dependent Fur repressor. When iron is limiting, the cell down-regulates sdh, possibly allowing Fe that would otherwise be used in assembling the succinate dehydrogenase enzyme to be available for essential functions. Other novel sRNAs positively and negatively regulate RpoS, while the function of many is not yet known.